Transformation is the integration into and expression of foreign DNA into a host cell. Other terms of art include DNA transfer or transfection of foreign DNA, followed by the stable incorporation of the DNA into a host cell genome. Because transformation can alter gene function, transformation can be utilized for gene therapy. Defective genes can be altered or corrected by the inactivation or replacement of mutant gene sequences. For example, expression of a gene sequence that allows the production of deleterious proteins may be curtailed by blocking the gene's promotor. Or, a defective gene sequence may be corrected by replacing it or by supplying missing bases.
Transgenic animals that have been transformed to carry human genes have become important models for in vivo study of human genetic disease. Gene defects can be established in these animals by inserting gene mutations, and then can be studied and treated by gene therapy. For example, recently developed transgenic mice carrying defective cystic fibrosus (CF) genes are being used as models that approximate human CF symptoms, and to study combinations of drugs that can be used for CF treatment (Dorin et al., 1992). In another research project, leukemic human hematopoiesis has been established in severe combined immunodeficiency, (SCID) mice to provide an in vivo model for the administration of therapeutic treatments for leukemia. (Ratajczak et al., 1992)
Gene therapy promises to become a technology of choice for treating human genetic diseases as more knowledge is gained of the molecular biology and pathology of genetic disorders, and as safe, efficacious gene transfer techniques are developed. As of October, 1992, thirty-seven gene therapy experiments had been approved worldwide. (Science 258:744) The National Institutes of Health have authorized a number of human gene therapy experiments in which a patient's own cells are transformed and returned to the patient to combat genetically derived disease. Among these experiments, adenosine deaminase (ADA) deficiency is being treated by using a retroviral vector to deliver and insert into the patients' white blood cells (which have been placed in culture) a normal ADA gene. Additional gene therapy trials underway include transformation of cells that have been taken from patients with ovarian cancer or cutaneous malignant melanoma. In one research project, genetically engineered viruses containing foreign genes are injected directly into patients' non-small-cell lung cancer tumors. At present "defective" (inactivated) viral vectors are used in the majority of gone therapy procedures. Somatic cell gone therapy has rather low efficiency, is not permanent, and usually must be repeated as, in time, transformed cells die or lose their effectiveness. A major goal of the number of researchers and biotechnology companies (including Systemics Corp.; CellPro, Inc.; Applied Immune Sciences, Inc.; Haemonetics Corp.; Baxter International; and Imclone Systems, Inc.) is the successful culture and transformation of human long-lived pluripotent or totipotent cells for sustained expression of introduced therapeutic genes (Dai et al., 1992).
Diagnosis of aberrant genetic conformation(s) and/or disease states in fixed tissues, cells, and nuclei isolated from cells can be accomplished by standard in situ hybridization (ISH) techniques for which there are numerous published protocols (Pinkel et al., 1986; Lawrence et al., 1989; Singer et al., 1986; Kallioniemi et al., 1992). Both isotopic and non-isotopic ISH have become important methods for identification of malignant cells and for human genome research. Although ISH results can be highly specific, commonly used ISH techniques have many disadvantages, including exacting fixation and storage requirements for individual cell and tissue types, lengthy probe incubation times, complex blocking, reporter processing and washing procedures, and tedious amplification protocols, as well as the need for access to state-of-the art microscopes and related equipment for analysis of results. In addition, high background sometimes resulting from the considerable manipulation (which may cause artifacts) of fixed cells during ISH treatments may make accurate interpretation of results difficult or impossible.
The method of the invention employs the specific catalytic activity of RecA protein (derived from the gram-negative prokaryote Escherischia coli) which was discovered about 28 years ago (Clark and Margulies, 1965). In in vitro homologous recombination assays it was learned that RecA protein can mediate both pairing and strand exchange between appropriate DNA molecules (for reviews see Kowalczykowski, 1991, Radding, 1989, 1991; also see Golub et al., 1992). More recently it was reported that in addition to DNA-DNA hybridization, RNA-DNA hybridization can be promoted by RecA protein: RecA protein coated single-stranded DNA (ssDNA) can recognize complimentarity with naked RNA (Kirkpatrick and Radding, 1992; Kirkpatrick et al., 1992).
Other proteins have been shown to catalyze strand-transfer reactions with results similar to RecA protein's, and it is anticipated that, among these prokaryotic proteins such as RecR from Bacillus subtilis (Alonso et al, 1993), and eukaryotic proteins such as STP from Saccharomyces cerevisiae (Hamatake et al., 1989), or RecA-like activity from S. cerevisiae (Kolodner et al., 1987), or Rec1 from Ustilago maydis (Kemic and Holloman, 1986) or RecA homolog in plastids in the plant Aradidopsis thaliana (Cerutti et al, 1992), or a protein from human cells which has homologous pairing activity (Moore and Fishel, 1990), or other proteins as yet undescribed may prove to be effective mediators of recombinant hybridization. One or more of these proteins may be found to function in pathways similar to RecA's and may provide catalytic activity which is analogous to RecA's for the purposes of this invention.
Although RecA has proved to be a useful in probing for a predetermined DNA sequence in the genome of a given cell, whether naturally occurring or introduced by recombinant techniques, its usefulness is largely limited to in vitro studies wherein the cells have already been fixed, and thus are no longer capable of reproduction. In one particular instance, RecA has been mentioned in connection with the use of viral capsids to introduce foreign DNA into a cell capable of being infected by the viral capsid. As set forth in detail in U.S. Pat. No. 4,950,599 to Bertling, polyoma or polyoma-like capsids are used to encapsulate an exogenous DNA sequence, followed by contacting the polyoma capsid to permissive cells, whereupon the exogenous DNA is taken up by the cell and exchanged with a substantially homologous DNA sequence already present in the cell. Bertling further mentions that the DNA should be combined with some sort of DNA binding protein to promote homologous recombination events, although such combination is not necessary to practicing the technique as broadly disclosed in the patent. Among the several such binding proteins mentioned is RecA. Unfortunately, the techniques discussed by Bertling rely on the use of an infecting vector. It will be understood by one skilled in the art that the construction of the infecting vector-DNA combination is a time-consuming, fairly labor intensive process that involves a significant loss of both convenience and time in order to achieve transformation of a given cell.
In view of techniques currently utilized for the detection of specific gene sequences in cells there is clear need for a simple and reliable in vivo method. There is also clearly a need for a simple, reliable method for in vivo transformation of living cells without excessive physical or chemical disruption of cell membranes, and which does not involve infectious agents or other vectors that may have a deleterious effect on the cells to be transformed.